For many human conditions, rats are superior to mice as model organisms, due to their physiology, responses to drugs, and performance during behavioral tests.

Their larger size facilitates molecular cell biology and physiology experimentation, particularly when the studies require dissection or isolation of specific cell types or specific organ structures. Therefore, rats are the preferred model organism for studies of human diseases including cancers such as breast, prostate, and bone marrow metastases.

However, the lack of convenient experimental tools to manipulate the rat genome has largely limited their use as genetic models for disease. Until recently, manipulation of the mouse genome by the introduction of genetically modified embryonic stem cells has given the mouse a distinct advantage over the rat as a model of many human diseases. Techniques such as ENU mutagenesis2 and mobile DNA technologies (transposons and retrotransposons)3 have produced knockout rat lines but are random and require intensive screening. The ZFN hybrid restriction enzyme combines the specificity of a zinc finger DNA binding motif with the nuclease activity of a Fok1 cleavage domain to create a double-stranded break at a single targeted site in the genome. Double-stranded breaks are repaired by non-homologous end-joining (NHEJ) with varying fidelity, introducing insertions or deletions at that site, and yielding mutations that cause the absence of the gene product.

With regard to cancer research, we have utilized this technique to disrupt exon 3 of the rat Tp53 gene, resulting in the creation of heterozygous (+/-) and homozygous null (-/-) rats. We show that the tissues of the knockout rats indeed express no Tp53 protein, and ongoing characterization will demonstrate the spectrum and incidence of tumor formation and validate them as models for studies of carcinogenicity, malignant pathogenesis, and testing of chemotheraputics. In the same capacity, we have created an immunocompromised model, lacking functional RAG1. This model may be used in the generation of xenografts for metastasis studies and bone marrow transplantation for cancer therapy, which will enable studies on drug delivery and metabolism in a more physiologically relevant model.

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